An interview with Rohit Sharma

In the October issue of the JCI, Sharma et al. demonstrate a novel mechanism by which pancreatic β cells proliferate. The authors found that rather than using a stem cell niche to expand, pancreatic β cells sense levels of insulin production and utilize the unfolded protein response (UPR) to proliferate. Their work demonstrates that moderate levels of endoplasmic reticulum (ER) stress drive β cell proliferation through the activation of activating transcription factor 6 (ATF6), thereby identifying potential druggable targets to improve treatment of patients with diabetes. This month, we interviewed first author Rohit Sharma and discussed the challenges in testing this novel hypothesis, the ways in which his past work informed his current understanding of the UPR pathway, and his advice for young trainees just beginning their research careers.

How do you foresee targeting the unfolded protein response pathway involved in pancreatic glucose regulation to impact diabetes?

It is known that chronic or continuous ER stress may cause β cell demise, resulting in type 2 diabetes (T2D). If we can reduce ER stress, that may benefit patients who are at high risk of developing T2D. In principle, we could also give short-term mild ER stress, and the individuals who are susceptible to T2D would have increased β cell mass, which may make them more resistant to diabetes later. But we need to test that; we are not there yet. We also found that overexpression of ATF6 in human islets results in increased β cell proliferation in vitro, so potentially therapies that activate ATF6 might be of use.

Were there any unexpected results or experiments that led you to pursue alternate paths in this work?

Yes. Our whole model was really unexpected. We started with the proteomic screen on the glucose-infused mice and found that many of the proteins that were altered were related to either UPR or ER-associated protein degradation. That itself was surprising to us, because it had been shown previously by others that ER stress actually results in β cell death. We found the complete opposite. That’s why we had to test each experiment by different methodologies to confirm that what we observed was actually right, and I think what we found is novel and could be impactful in the field of diabetes.

What was the reaction from your peers when you first started presenting these results to them?

When we were presenting this work, there was initially a lot of resistance to our hypothesis. But it really all makes sense, since more than 50% of the total protein in β cells is insulin. Whenever there is demand, rather than sensing signals from other tissues, β cell UPR senses insulin demand and expands β cells to meet demand. Also, a lot of effort has been put forth to find stem cells in the pancreas for β cell regeneration, but nobody could show convincingly that new β cells are generated from stem cells. Work by others has shown that new β cells arise from preexisting β cells, but here we have additionally shown that β cell UPR senses the increased insulin demand to increase β cell number.

In what ways did your graduate training provide the expertise needed to succeed during your postdoctoral fellowship?

My PhD work under the mentorship of Dr. Manni Luthra-Guptasarma was on protein misfolding, specifically HLA-B27, which is known to be associated with ankylosing spondylitis. All of my work there was trying to figure out how protein misfolding results in that disease. We found that misfolded HLA-B27 was also present on the cell surface in patients with ankylosing spondylitis, along with correctly folded MHC complex, but as an aggregate. I had a lot of experience in biophysical techniques there, where we were studying ER stress as well, which helped me with this project. In between, I have worked in different fields. In my first postdoc at the University of Texas Southwestern Medical Center in Dallas under the mentorship of Dr. Joel Taurog, I worked in the same field, HLA-B27 misfolding and spondyloarthropathies. However, due to funding, I had to move to the University of Pittsburgh, where I worked in the cancer field. In cancer cells, it is the opposite of β cells, as cancer cells are highly proliferative, so I was studying why they were highly proliferative and analyzing the function of RNA-editing enzymes in cancer cells and normal cells. My PhD and my previous postdocs were in protein misfolding and proliferation, respectively. When I joined this lab, the proteomic screen was already performed to find novel targets for β cell proliferation. When they told me what they found, I jumped on this project, and I think that I was lucky to choose this project to work on and obtain the results we found.

What has been the role of mentorship in facilitating your career development thus far?

All through the years, all of my mentors were really encouraging and supportive to me. I had to switch fields because of funding issues. Since 2008 when NIH funding began to decrease, two of my three previous labs were shut down because of funding issues. My mentors and my productivity in terms of publications from each lab really helped me to get to the next position. Mentorship is the most important aspect, since I received really good training in different fields. The questions were similar, but the fields were completely different.

What have been the most significant challenges that you have faced as a postdoctoral fellow?

For me, it was funding every time. But since I joined Dr. Alonso’s lab, which had recently received NIH funding, it has been much easier for me to work. In this field of diabetes research, specifically in β cell proliferation, the most challenging aspect is the material, which is really limiting. For example, from a mouse, you only get 200 to 250 islet equivalents, from which you can do only one well of a Western blot, which makes working on primary mouse β cells much more expensive. Cell lines are not ideal for proliferation studies, as proliferation rates are much higher in cell lines as compared to primary mouse or human β cells. This makes the field of β cell proliferation really challenging.

Regarding funding, what were the particular challenges that you faced, and have you found any solutions so far?

During my first three years, I changed fields every year, so my publications from each lab were in different fields, which made it harder to apply for postdoctoral funding. I now have a few papers in the field of diabetes, including this JCI paper, which will help me tremendously in my career and to obtain funding.

What advice would you offer trainees who have faced similar personal and professional challenges?

I would advise them to have patience, persistence, and keep working hard, because hard work never gets wasted. In my case, even when I was switching fields, I was working hard and was able to produce publications from each lab, which helped me get the next position. I definitely want to advise new graduates that they should choose the lab wisely as well as the field of research, as both may affect their success.

What were the main differences between training in India versus here in terms of the work environment?

Over here, it is much easier to work, because reagents are readily available. In India, for each experiment, you have to plan a month or two in advance, even for a small thing such as an antibody or oligos. Even for basic instruments for developing a Western blot, one had to go to a different lab. In my field of protein folding/misfolding, we were using techniques of chromatography, fluorescence, and circular dichroism. To do that, we had to go to a different institute, sometimes in a different city. Over here, everything is really well connected, and you have cores in every institution. I should note though that the situation in India has changed since I moved from there, as the Indian government is now spending lot of money in the field of technology and research.

In the future, what role do you think basic science or translational research will play in your career?

I think basic science is very important to understanding the disease mechanisms, but at the same time, translational research is equally important in actually treating the disease. As one of my mentors from Pittsburgh used to say, we need research not to treat mouse diabetes, but to treat human diabetes. We definitely want to contribute to a better understanding of this disease and treatment of humans to make life better for diabetes patients. So translational science will definitely play a very important role in my career.

About the First Author

Rohit Sharma, PhD, is currently a postdoctoral researcher at the University of Massachusetts Medical School in Laura Alonso’s laboratory. He completed his bachelor’s and master’s degrees at Panjab University in India and received his PhD in 2008 from the Postgraduate Institute for Medical Education and Research (PGIMER) in Chandigarh, India, under the mentorship of Manni Luthra-Guptasarma.

About the interviewers

Freddy T. Nguyen is an MD/PhD candidate at the University of Illinois at Urbana-Champaign. He is the founder of the American Physician Scientists Association and served on the Associate Member Council of the American Association for Cancer Research. His research interests currently lie at the intersection of biomedical optics and cancer research. He received his BS in chemistry and BA in mathematics from Rice University.

Evan Noch, MD, PhD, is a third-year resident in a research track position within the Department of Neurology at Weill Cornell Medical Center–New York Presbyterian Hospital in New York City. He currently serves as the Chief Resident for Research for the program. He studies glioblastoma metabolism in the lab of Lewis Cantley and plans to pursue a fellowship in neuro-oncology. Dr. Noch received his MD and PhD from the MD/PhD program at Temple University School of Medicine.

Abstract

Although stem cell populations mediate regeneration of rapid turnover tissues, such as skin, blood, and gut, a stem cell reservoir has not been identified for some slower turnover tissues, such as the pancreatic islet. Despite lacking identifiable stem cells, murine pancreatic β cell number expands in response to an increase in insulin demand. Lineage tracing shows that new β cells are generated from proliferation of mature, differentiated β cells; however, the mechanism by which these mature cells sense systemic insulin demand and initiate a proliferative response remains unknown. Here, we identified the β cell unfolded protein response (UPR), which senses insulin production, as a regulator of β cell proliferation. Using genetic and physiologic models, we determined that among the population of β cells, those with an active UPR are more likely to proliferate. Moreover, subthreshold endoplasmic reticulum stress (ER stress) drove insulin demand–induced β cell proliferation, through activation of ATF6. We also confirmed that the UPR regulates proliferation of human β cells, suggesting that therapeutic UPR modulation has potential to expand β cell mass in people at risk for diabetes. Together, this work defines a stem cell–independent model of tissue homeostasis, in which differentiated secretory cells use the UPR sensor to adapt organ size to meet demand.